2 research outputs found
Aminoacyl-tRNA Substrate and Enzyme Backbone Atoms Contribute to Translational Quality Control by YbaK
Amino acids are covalently attached to their corresponding
transfer
RNAs (tRNAs) by aminoacyl-tRNA synthetases. Proofreading mechanisms
exist to ensure that high fidelity is maintained in this key step
in protein synthesis. Prolyl-tRNA synthetase (ProRS) can misacylate
cognate tRNA<sup>Pro</sup> with Ala and Cys. The <i>cis</i>-editing domain of ProRS (INS) hydrolyzes Ala-tRNA<sup>Pro</sup>,
whereas Cys-tRNA<sup>Pro</sup> is hydrolyzed by a single domain editing
protein, YbaK, <i>in trans</i>. Previous studies have proposed
a model of substrate-binding by bacterial YbaK and elucidated a substrate-assisted
mechanism of catalysis. However, the microscopic steps in this mechanism
have not been investigated. In this work, we carried out biochemical
experiments together with a detailed hybrid quantum mechanics/molecular
mechanics study to investigate the mechanism of catalysis by Escherichia coli YbaK. The results support a mechanism
wherein cyclization of the substrate Cys results in cleavage of the
Cys-tRNA ester bond. Protein side chains do not play a significant
role in YbaK catalysis. Instead, protein backbone atoms play crucial
roles in stabilizing the transition state, while the product is stabilized
by the 2′-OH of the tRNA
Substrate and Enzyme Functional Groups Contribute to Translational Quality Control by Bacterial Prolyl-tRNA Synthetase
Aminoacyl-tRNA synthetases activate specific amino acid
substrates
and attach them via an ester linkage to cognate tRNA molecules. In
addition to cognate proline, prolyl-tRNA synthetase (ProRS) can activate
cysteine and alanine and misacylate tRNA<sup>Pro</sup>. Editing of
the misacylated aminoacyl-tRNA is required for error-free protein
synthesis. An editing domain (INS) appended to bacterial ProRS selectively
hydrolyzes Ala-tRNA<sup>Pro</sup>, whereas Cys-tRNA<sup>Pro</sup> is
cleared by a freestanding editing domain, YbaK, through a unique mechanism
involving substrate sulfhydryl chemistry. The detailed mechanism of
catalysis by INS is currently unknown. To understand the alanine specificity
and mechanism of catalysis by INS, we have explored several possible
mechanisms of Ala-tRNA<sup>Pro</sup> deacylation via hybrid QM/MM
calculations. Experimental studies were also performed to test the
role of several residues in the INS active site as well as various
substrate functional groups in catalysis. Our results support a critical
role for the tRNA 2′-OH group in substrate binding and catalytic
water activation. A role is also proposed for the protein’s
conserved GXXXP loop in transition state stabilization and for the
main chain atoms of Gly261 in a proton relay that contributes substantially
to catalysis